‘Transgenics’ appear to be the ‘need of the hour’ in view of the ever growing demand for quality crops all over the world (Somerville C. R. 1994. In: Production and Uses of Genetically Transformed Plants, Chapman and Hall, London (eds. Bevan M. W., Harrison R. D., Leaver C. J.).
Transgenic production through genetic transformation of plants is brought about by introducing desired gene(s) from any living organism into the concerned plant by different methods of DNA delivery in order to impart different traits for crop improvement viz. disease and pest resistance, longer shelf life, better and added nutritional value, varied colours and scents, tolerance to abiotic stresses like drought, flood, cold, salt etc.
Of the different methods of gene delivery into plants, the most popular and cost effective method is producing transgenics through Agrobacterium tumefaciens. By this method explants that have the ability to regenerate into full grown plants under tissue culture conditions are infected with the disarmed and engineered strains of the soil bacteria ‘Agrobacterium tumefaciens’ containing the desired engineered genes.
Once the infection has occurred, the virulence genes of the Agrobacterium tumefaciens gets triggered and this enables the bacterium to pass on the T-DNA containing the desired engineered gene into the plant cell/nucleus and finally the desired engineered gene gets integrated into the plant genome which then becomes genetically transformed.
For successful and high frequency of genetic transformation of plants, fresh actively growing cultures of Agrobacterium tumefaciens containing the engineered and disarmed T-DNA is generally grown for 12–16 hr at 25–30° C. and 150–200 rpm in dark upto its log phase of growth when the cell density becomes 1×109 cells/ml. The explants are then infected with these fresh cultures and allowed to incubate for a few days at optimal temperature and pH so that the T-DNA with the desired engineered gene is passed on to plant cell/nucleus which finally gets integrated into the plant genome. Once the gene is integrated either transiently or stably into plant genome, the residual Agrobacterium tumefaciens needs to be eliminated totally or else the residual Agrobacterium overgrows on the explant and prevents it from regenerating into a healthy plant and in severe cases even kills the explants.
A serious drawback of Agrobacterium tumefaciens mediated genetic transformation protocols is the requirement of high cost and labour intensive steps of washing and subsequent repeated culturing of transformants onto a medium containing expensive bactericidal antibiotics in order to kill Agrobacterium tumefaciens. These repeated washings and sub-culture involving bactericidal antibiotics not only result in vitrification of the transformants but also in their loss due to severe bacterial overgrowth. The problem is so severe that about 50% of the transformants are lost due to bacterial overgrowth and many a times, the generally employed bactericidal antibiotics fail to prevent this loss at feasible doses.
In a preliminary study, crude leaf extracts were found to have a bactericidal effect on the overgrowing Agrobacterium tumefaciens after co-cultivation, yet another important problem that was encountered was the loss and death of transformants due to phenol oxidation. It was therefore, necessary to optimize a concentration or composition of tea leaf extract that would not only kill the residual Agrobacterium tumefaciens after the transfer and integration of its T-DNA into the plant genome but would also overcome the problem of loss and death of the explants due to phenol oxidation.
Tea leaves contain high levels of polyphenols which are composed of 6 types of catechins (C) and their derivatives viz., epicatechin (EC), gallocatechins (GC), epigallocatechins (EGC), epicatechin gallate (ECg), epigallocatechin gallate (EGCg). Besides polyphenols, tea leaves also contain caffeine, amino acids and other nitrogenous compounds, vitamins, inorganic elements, carbohydrates and lipids (Chu D-C and Juneja L. R. General chemical composition of green tea and its infusion. In: Chemistry and Application of Green Tea. 1997. CRC Press, N.York. eds. Yamamoto T., Juneja L. R., Chu D-C, Kim M.). Tea polyphenols are known to show several biochemical activities such as inhibition of bacterial mutation (Kada T., Kaneka K., Matsuzaki T. and Hara Y. 1985. Detection and chemical identification of natural biomutagens. A case of green tea factor. Mutation Research 150: 127), inhibition of HIV reverse transcriptase activity (Nokane H. and Ono K. 1990. Differential inhibitory effects of some catechins derivatives on the activities of human immunodeficiency virus reverse transcriptase and cellular deoxyribonucleic and ribonucleic acid polymerases, Biochemistry 29: 2841–5).
Tea polyphenols have shown anticaries effects by inhibiting the adherence of P. gingivalis onto buccal epithelial cells at concentrations 250–500 μg/ml which is much larger compared to the effective concentrations of antibiotics (Sakanaka S., Aizawa M., Kim M. and Yamamoto T. 1996 Inhibitory effects of green tea polyphenols on growth and cellular adherence of an oral bacterium Porphyromonal gingivalis. Biosci. Biotechnol. Biochem. 60: 745; Sakanaka S. 1997. Green tea polyphenols for prevention of dental caries In: Chemistry and Application of Green Tea. eds. Yamamoto T., Juneja L. R., Chu D-C, Kim M.). Tea polyphenols have also been reported to possess antiviral activity dependent on the galloyl moiety linked by ester linkage in catechin molecule (John T. J. and Mukundan P. 1979.
Virus inhibition by tea, caffeine and tannic acid. Indian J. Med. Res. 69: 542; Nakayama M., Toda M., Okubo S. and Shimamura T. 1990. Inhibition of influenza virus infection by tea. Lett. Appl. Microbiol. 11: 38). Huang and Frankel, 1997 (Huang S-W and Frankel E. N. 1997 Antioxidant activity of tea catechins in different lipid systems. J. Agric. Food Chem. 45: 3033–8) have reported antioxidant activity of tea catechins in liposomes. Also tea catechins have been reported to scavenge the oxidative radicals that cause damage to the DNA (Yen G-W. and Chen H-Y. 1995. Antioxidant activity of various tea extracts in relation to their antimutagenecity. J. Agric. Food Chem. 43: 27–32), and their oxygen radical absorbing action with antiproliferative action in human epidermoid carcinoma A431 cells (Lin Y-L., Juan I-M., Chen Y-L., Liang Y-C. and Lin J-K. 1996. Composition of polyphenols in fresh tea leaves and associations of their oxygen-radical-absorbing capacity with antiproliferative actions in fibroblast cells. J. Agric. Food Chem. 44: 1387–94).
Saeki et al. 1999 (Saeki K., Sano M., Miyase T., Nakamura Y., Hara Y., Aoyagi Y. and Isemura M. Apoptosis-inducing activity of polyphenol compounds derived from tea catechins in human histiolytic lymphoma U937 cells. Biosci. Biotechnol. Biochem. 63: 585–7) have recorded the apoptosis inducing activity of polyphenols of tea as evidenced by inhibition of DNA ladder formation and chromatin condensation in human histolytic lymphoma cells. Antimicrobial activity of green tea extract has also been well documented at minimum inhibitory concentration or MIC (250 μg/ml to 1000 μg/ml) for several micro-organisms that are harmful to human health (In: Chemistry and Application of Green Tea. 1997. CRC Press, New York. eds. Yamamoto T., Juneja L. R., Chu D-C, Kim M.). Crude tea leaf extract thus, appears to be a potent agent for bactericidal formulations during genetic transformation, once the problem of phenolic oxidation and hence death of the transformants is alleviated.
One of the problems encountered during plant tissue transformation with A. tumefaciens is the effective elimination of the bacterium after transfer of the transgenes has taken place. When using Agrobacterium as a tool in plant genetic engineering, there is a risk that if not all bacteria are eliminated after transformation, the residual bacteria will kill the transgenic plants. Therefore, strategies in genetic transformation generally require a balance between the bactericidal activity and the normal morphogenetic response of transformed tissue. Different reports on these problems include the report by Colby and Meredith (Colby S. M. and Meredith C. P. Kanamycin sensitivity of cultured tissues of Vitis Plant Cell Reports 9(5): 237–40) who used the bactericidal antibiotic carbenicillin for killing residual Agrobacterium tumefaciens during genetic transformation but the drawback of their report was that carbenicillin had inhibitory effects on plant regeneration which sometimes resembled those of growth inhibitors.
Also, Park et al., 1990 (Park Y. G., Shin D. W., Kim J. H. 1990. Journal of Korean Forestry Society 79(3): 278–84) used a combination of cefotaxime (200 mg/liter) and carbenicillin (300 mg/liter) for Agrobacterium mediated transformation of Populus nigra X P. maximowiczii leaves. The drawback of the protocol was that although, the growth of Agrobacterium tumefaciens strain 6044 was reduced, yet, the residual Agrobacterium tumefaciens was not totally eliminated and the rate of regeneration was also as low as 10%.
Yurkova et al., 1993 (Yurkova G. N., Chugunkova T. V. and Shevtsov I. A. 1993. Effect of antibiotics on the tissue culture of sugarbeet and fodder beet. Tsitologiya-i-Genetika 27(2): 3–6) used a combination of claforan and carbenicillin as inhibitors of Agrobacterium cells during transformation of diploid sugarbeet hybrid and 2 diploid varieties of fodder beet. However, the drawback of this report was that this combination had an inhibiting effect on shoot formation.
Yepes and Aldwinckle, 1994 (Yepes L. M. and Aldwinckle H. S. 1994. Micropropagation of thirteen Malus cultivars and rootstocks, and effect of antibiotics on proliferation. Plant Growth Regulation 15(1): 55–67) used cefotaxime at 200 mg/liter and carbenicillin at 500 mg/liter for the transformation of 13 apple cultivars. However, the drawback of the report was that while cefatoxime caused abnormal shoot morphology, carbenicillin alone or in combination with cefotaxime at 200 mg/liter, inhibited proliferation and caused excessive enlargement of the basal leaves, inducing callus formation and release of phenolic compounds in the medium. Lin et al., 1995 (Lin-J J., Assad-Garcia N. and Kuo, J. 1995.
Plant hormone effect of antibiotics on the transformation efficiency of plant tissues by Agrobacterium tumefaciens cells. Plant Science Limerick. 109(2): 171–177) reported the use of 250 to 2000 μg/ml carbenicillin for the inhibition of bacterial (A. tumefaciens strains, LBA4404, C58 and EHA101) growth in tobacco leaf explants wherein they showed that LBA4404 was the most sensitive to carbenicillin and cefotaxime. However, the drawback of the protocol was that the regeneration efficiency from leaf explants decreased with the addition of increasing concentration of carbenicillin on MS medium containing 0.5 μg/ml of benzyladenine and/or 2,4-D wherein they emphasized on the toxic effects on leaf explants when grown on MS medium containing a combination of 250 μg/ml carbenicillin and 1 μg/ml 2,4-D due to the auxin related chemical structures of 2,4-D or NAA and carbenicillin. The major drawback of this protocol is that such plant growth regulators (2,4-D, BA or NAA) are generally used in most regeneration media that are employed for Agrobacterium tumefaciens mediated transformation.
Sarma et al, 1995 (Sarma K. S., Evans N. E. and Selby C. 1995. Effect of carbenicillin and cefotaxime on somatic embryogenesis of Sitka spruce (Picea sitchensis (Bong.) Carr. Journal of Experimental Botany 46(292):, 1779–81) reported the use of antibiotics viz. carbenicillin and cefotaximethese in an attempt to transform Sitka spruce somatic embryos using Agrobacterium based vectors. They reported that carbenicillin should not be used for transformation of Sitka spruce. The drawback of the report was that carbenicillin prevented the development of mature somatic embryos, reduced early stage embryos by >90% and tissue growth by 50%.
Cefotaxime had no effect on overall tissue growth, but reduced the development of early and mature embryos by 20% and 66–80%, respectively.
Shackelford and Chlan 1996 (Shackelford N. J. and Chlan C. A. 1996. Identification of antibiotics that are effective in eliminating Agrobacterium tumefaciens. Plant Molecular Biology Reporter 14(1): 50–5) performed an assay using ten antibiotics viz. cefotaxime, carbenicillin, erythromycin, spectinomycin, polymixin B, chloramphenicol, methicillin, augmentin 500, augmentin 250 and moxalactam [latamoxef] inorder to identify the antibiotics that are most effective against A. tumefaciens strains EHA101 and LBA4404, and to determine if these antibiotics inhibited tobacco callus and shoot formation. This report indicates that a major drawback of most transformation protocol is time consuming assays involving several costly antibiotics for the effective elimination of A. tumefaciens after gene transfer into plant explant has taken place.
Barrett et al., 1997 (Barrett C., Cobb E., McNicol R. and Lyon G. 1997 A risk assessment study of plant genetic transformation using Agrobacterium and implications for analysis of transgenic plants. Plant Cell Tissue and Organ Culture. 47(2): 135–44) examined Agrobacterium transformation systems for Brassica, Solanum and Rubus, using carbenicillin, cefotaxime and ticaracillin [ticarcillin], respectively, to eliminate contamination, for the presence of residual Agrobacterium. Results indicated that none of the antibiotics tested succeeded in eliminating Agrobacterium and the contamination levels increased in explants from 12 to 16 weeks to such an extent that Solanum cultures senesced and died. This may be due to the fact that in some cases the minimum bactericidal concentration values (concentration to be used for elimination of contaminants in culture) for the three antibiotics were higher than the concentrations employed in the culture medium.
Moreover, even up to 6 months after transformation, 50% of contaminated material still harboured bacterial cells with the binary vector at levels of 107 colony forming units per gram. This report indicates a major drawback with most of the generally used antibiotics are ineffective for Agrobacterium elimination in plants like Brassica, Solanum and Rubus and affects their regeneration as well as survival efficiency.
Hammerschlag et al., 1997. (Hammerschlag F. A., Zimmerman R. H. Yadava, U. L., Hunsucker S. and Gercheva P. 1997. Effect of antibiotics and exposure to an acidified medium on the elimination of Agrobacterium tumefaciens from apple leaf explants and on shoot regeneration. Journal of the American Society for Horticultural Science. 122(6): 758–63) tested a range of antibiotics and their short-term exposure to an acidified (pH 3.0) medium for their effects on eliminating A. tumefaciens, supervirulent strain EHA101 (pEHA101/pGT100), on leaf explants of Royal Gala apples (Malus domestica [M. pumila]) and on shoot regeneration.
They reported that the exposure of leaf explants to regeneration and elongation media containing 100 μg/ml of the antibiotics carbenicillin (crb), cefotaxime (cef), and cefoxitin (mefoxin (mef)), singly or in combination for 52 days, did not eliminate A. tumefaciens from the explants. The drawback of this report was that even short-term (1 to 18-hour) vacuum infiltration with 500 μg/ml of any of the antibiotics did not inhibit regeneration and failed to completely eliminate A. tumefaciens from leaf explants.
Cheng et al., 1998 (Cheng, Z. M., Schnurr, J. A. Kapaun, J. A. 1998. Timentin as an alternative antibiotic for suppression of Agrobacterium tumefaciens in genetic transformation. Plant Cell Reports 17(8): 646–9) studied the effects of timentin on shoot regeneration of tobacco (Nicotiana tabacum) and Siberian elm (Ulmus pumila) and its use for the suppression of Agrobacterium tumefaciens during genetic transformation. Timentin—a mixture of ticarcillin and clavulanic acid was used effectively at concentrations of 200–500 mg/l and at ratios of ticarcillin:clavulanic acid at 50:1 and 100:1. The drawback of this report is that although timentin is a less expensive alternative to the use of carbenicillin or cefotaxime antibiotics for the effective suppression of A. tumefaciens after genetic transformation yet it is expensive synthetic antibiotic as compared to natural agents like crude tea extracts.
Ling et al., 1998 (Ling H. Q., Kriseleit D. and Ganal M. W. 1998. Effect of ticarcillin/potassium clavulanate on callus growth and shoot regeneration in Agrobacterium -mediated transformation of tomato (Lycopersicon esculentum Mill.) Plant Cell Reports 17(11): 843–7) studied the effect of Cefotaxime, ticarcillin/potassium clavulanate on tomato transformation. Cefotaxime did not inhibit callus growth in culture medium, but it clearly decreased shoot differentiation. While cefatoxime showed a strong negative effect on callus growth, shoot regeneration and transformation efficiency, the ticarcillin/potassium clavulanate was more economical and effective than carbenicillin and cefotaxime. The drawback of the report is that although ticarcillin/potassium clavulanate was a very good alternative to eliminate Agrobacterium tumefaciens in plant transformations yet it is an expensive synthetic antibiotic as compared to natural agents like crude tea extracts.
Joersbo et al. 2000 (Joersbo M., Brunstedt, J. Marcussen, J. Okkels F. T. 2000. Transformation of the endospermous legume guar (Cyamopsis tetragonoloba L.) and analysis of transgene transmission. Molecular-Breeding 5(6): 521–9) used carbenicillin and cefotaxime, for the elimination of Agrobacterium after co-culture, wherein they found considerable toxicity was displayed to guar tissues due to beta-lactams. When the beta-lactams were replaced by the non-phytotoxic beta-lactamase inhibitor “sulbactam” alongwith thidiazuron and silver thiosulfate the problem was overcome. The drawback of this report was that while the commonly used carbenicillin and cefotaxime were phytotoxic to the explants used, replacement of beta-lactams by sulbactam is a costly process.